1. Field of the Invention
The present invention is directed to novel composite plasmids for use as cloning vehicles in Coryneforms such as Rhodococcus and Corynebacterium, and also in E. coli, Bacillus subtilis, and Staphylococcus aureus. The composite plasmids of the present invention are capable of replicating in Rhodococcus equi and also capable of replicating in other Coryneforms, E. coli, Bacillus subtilis and Staphylococcus aureus. In addition the composite plasmids include selectable markers, in one instance genes for resistance to antibiotics, in another instance the lacZ gene. The selectable markers may be employed to assign the marker phenotype to a host microorganism. Further, a novel transposon delivery vehicle is disclosed.
2. Background of Related Information
Coryneforms constitute a broad morphological group of bacteria which are widely distributed in nature. The Coryneform group of bacteria are gram-positive microorganisms having a rod or club-like shape. The group is composed of several different genera, the most widely known of which is the Corynebacteria. The Coryneforms are both agriculturally and industrially important microorganisms. Many species inhabit the soil and cause disease in plants such as alfalfa, bean, beet, corn, tomato, potato, wheat and sugar cane. In addition, certain species of the Coryneforms are used industrially in the production of amino acids and in steroid conversions Other strains of Coryneforms have been found to utilize hydrocarbons, and to synthesize emulsifying agents and antitumor agents.
Recombinant DNA plasmids capable of functioning as shuttle vectors in CorYnebacteria have been described previously. Miwa, K. et al., U.S. Pat. No. 4,514,520, disclose Coryneform shuttle vectors capable of being propagated in Corynebacteria/E. coli or Corynebacteria/B. subtilis. In addition, these shuttle vectors have the capacity to confer cellular resistance to antibiotics. Miwa, et al. further disclose techniques for plasmid manipulation, transformation, and expression.
Ozaki, A. et al., Mol. Gen. Genet. 196:175-178 (1984), disclose the construction of a shuttle vector capable of replication in both Corynebacterium glutamicum and E. coli. The plasmid conferred cellular resistance to kanamycin, tetracycline, ampicillin, and chloramphenicol.
Yoshihama, M. et al., J. Bacteriol. 162:591-597 (1985), disclose a Coryneform shuttle vector capable of replicating in both Corynebacterium glutamicum and Bacillus subtilis. This reference further discloses a transformation protocol resulting in approximately 10.sup.4 transformants per ug of plasmid DNA.
European Patent Application 0 088 166 A2 discloses plasmid vectors autonomously replicable in the microorganisms belonging to the genus Corynebacterium or Brevibacterium and having appropriate selectable markers and suitable cloning sites These plasmid vectors are utilized to transform a host microorganism selected from the genus Corynebacterium or Brevibacterium, wherein a DNA fragment containing at least one gene is inserted into a vector DNA, and at least one of both DNAs is foreign to the host microorganism.
Katsumata, R. et al., U.S. Pat. No. 4,500,640, disclose plasmids capable of replicating autonomously in a microorganism belonging to the genus Corynebacterium or Brevibacterium and which carry a gene for resistance to streptomycin and/or spectinomycin. The plasmids are disclosed as useful as cloning vectors. Katsumata, R. et al., U.S. Pat. No. 4,489,160, disclose a plasmid obtained from Corynebacterium glutamicum containing multiple restriction endonucleases cleavage sites. The plasmids are disclosed as useful for producing vectors for microorganisms belonging to the genus Corynebacterium.
In spite of the recent advances in vector technology, a need has continued to exist for additional shuttle vectors capable of replication in multiple host microorganisms, these shuttle vectors containing heritable, selectable markers.